Comprehensive Identification of the Genetic Requirements for ESX-1 Secretion

描述（由申请人提供）：ESX-1 （ESAT-6系统1）分泌系统是分枝杆菌和革兰氏阳性病原体的关键毒力决定因素。由于ESX-1功能所需的所有基因尚未被确定，因此在理解该分泌系统的分子机制方面存在根本性的空白。缺乏这一信息是因为由于ESX-1功能的直接、高通量测量的技术限制，从未进行过ESX-1分泌需求的饱和遗传筛选。然而，获得这些知识对于分枝杆菌毒力的基本理解至关重要。长期目标是了解分枝杆菌发病机制下蛋白质分泌系统的基本分子机制。本提案的目的是通过鉴定致病性分枝杆菌中ESX-1分泌所需的所有基因，实现实现这一目标的第一步。假设这可以通过直接饱和遗传筛选来监测ESX-1功能来实现。一种基于蛋白质组学的方法允许直接，高通量监测分枝杆菌ESX-1底物的分泌，并在申请人的实验室中设计和验证。这一建议的基本原理是，在完成该项目中积累的知识将极大地促进对这一重要毒力途径的理解。因此，提出了以下具体目标：进行直接的、饱和的遗传筛选，全面鉴定致病性分枝杆菌中ESX-1分泌系统的成分。利用全菌落MALDI-TOF（基质辅助激光解吸电离飞行时间）质谱法直接监测细菌中ESX-1蛋白分泌的方法已经在申请人手中建立起来。该方法将用于对海洋分枝杆菌转座子文库进行大规模筛选，以鉴定不分泌ESX-1底物的菌落。提议的方法是创新的，因为它代表了对现状的直接背离，并将克服当前识别ESX-1系统所有组件的技术障碍。这一贡献将是重要的，因为它有望在理解ESX-1分泌的基本生物学方面取得巨大进展。

DESCRIPTION (provided by applicant): The ESX-1 (ESAT-6 system 1) secretion system is a key virulence determinant in both mycobacteria and Gram-positive pathogens. There is a fundamental gap in understanding the molecular mechanisms of this secretion system because all of the genes required for ESX-1 function have not been identified. The absence of this information is because a saturating genetic screen for requirements of ESX-1 secretion has never been performed due to technical limitations in the direct, high throughput measurement of ESX-1 function. However, the acquisition of such knowledge is critical to the basic understanding of mycobacterial virulence. The long-term goal is to understand the basic molecular mechanisms of protein secretion systems that underlie mycobacterial pathogenesis. The objective of this proposal is to attain the primary step toward this goal by identifying all of the genes required for ESX-1 secretion in pathogenic mycobacteria. The hypothesis is that this can be accomplished using a direct saturating genetic screen to monitor ESX-1 function. A proteomics based method that allows direct, high throughput monitoring of secretion of mycobacterial ESX-1 substrates was devised and validated in the applicant's laboratory. The rationale that underlies this proposal is that the knowledge amassed in the completion of this project will greatly advance the understanding of this important virulence pathway. Therefore, the following specific aim has been proposed: To conduct a direct, saturating genetic screen to comprehensively identify the components of the ESX-1 secretion system in pathogenic mycobacteria. A method using whole colony MALDI-TOF (matrix assisted laser desorption ionization-time of flight) mass spectrometry to directly monitor ESX-1 protein secretion in bacteria has been established as feasible in the applicant's hands. This method will be used to conduct large scale screening of a Mycobacterium marinum transposon library to identify colonies that do not secrete ESX-1 substrates. The proposed approach is innovative because it represents a direct departure from the status quo and will overcome the current technical hurdle to identifying all components of the ESX-1 system. This contribution will be significant because it is expected to result in a great advance in understanding the basic biology of ESX-1 secretion. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because understanding the basic biology of how mycobacterial pathogens cause disease is the first step in developing new anti- Tuberculosis diagnostics and therapeutics, which would help alleviate the current Tuberculosis epidemic. This research is relevant to the part of NIH's mission that relates to the pursuit of fundamental knowledge that will lead to the development of scientific resources that will assist in disease prevention.